Western Blotting for DNA Damage Signaling

The standard western blotting protocol was conducted to detect the levels of indicated proteins as described previously (Yuan et al, 2017 (link); Li et al, 2021 (link)). Antibody against GAPDH (AF0006) was from Beyotime (Shanghai, China). Antibodies against γ‐H2AX (#2577), CDT1 (#8064), Chk1 (#2360), p‐Chk1(S317, #2344), p‐Chk2 (Thr68, #2661), caspase‐3 (#9662), caspase‐7 (#12827), caspase 9 (#9502), PARP (#9542), Bak (#12105), BID (#2002), Puma (#4976), Noxa (#14766), Bcl‐XL (#2764), XIAP (#14334), MRE11 (#4895), c‐IAP1 (#7943), c‐IAP2 (#3130), STAT1 (#14994), p‐STAT1 (Tyr701, #9167), p‐STAT1 (Ser727, #8826), STAT3 (#9139), p‐STAT3 (Ser727, #9145), STING (#13647), p‐STING (#19781), TBK1 (#3504), p‐TBK1 (#5483), IRF3 (#11904), p‐IRF3 (Ser386, #37829), p38 (#9212), p‐p38 (#9211), ERK1/2 (#9102), p‐ERK1/2 (#4370), MEK1/2 (#4694), p‐MEK1/2 (#9154), JNK (#9252), Axin2 (#2151), KU70 (#4588) and KU80 (#2753) were from Cell Signaling Technology. Antibodies against RPA32 (sc‐271578), Bax (sc‐493), MCL1 (sc‐819), PTIP (sc‐367459), Chk2 (sc‐9604), PARP2 (sc‐30622), XRCC1 (sc‐11429), XRCC3 (sc‐271714), MLH1 (sc‐581), MSH2 (sc‐494), TNKS1/2 (sc‐365897), p‐JNK (sc‐6254), PARP1 (sc‐7150) and PAR [pADPr (10H) (sc‐56198)] were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against p‐RPA32 (PLA0071) was from Sigma (Shanghai, China). Antibody against RAD51 (ab63801), CTIP (ab70163), MCRS1/MSP58 (ab247013) and PALB2 (ab202970) were from Abcam. Antibody against MAD2L2/REV7 (BD‐612266) was from BD Biosciences. Antibody against BRCA1 (OP92) and BRCA2 (OP95) were from Millipore. Goat anti‐mouse IgG horseradish peroxidase antibody was provided by Merk/Calbiochem (Darmstadt, Germany). All of the primary antibodies, except for GAPDH, were used after 1:1,000 dilution, and GAPDH primary antibody was used after 1:5,000 dilution. Second antibodies were used following 1:2,000 dilution.

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Wang L., Wang P., Chen X., Yang H., Song S., Song Z., Jia L., Chen H., Bao X., Guo N., Huan X., Xi Y., Shen Y., Yang X., Su Y., Sun Y., Gao Y., Chen Y., Ding J., Lang J., Miao Z., Zhang A, & He J. (2023). Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance. EMBO Molecular Medicine, 15(3), e16235.

Publication 2023

Anti antibody Antibodies Antibody Axin2 Brca1 Brca2 C iap1 C iap2 Caspase 3 Caspase 7 Caspase 9 Ctip Dilution Erk1 Gapdh Goat Igg horseradish peroxidase Irf3 Ku70 Mcl1 Mek1 Mlh1 Mouse Palb2 Parp1 Parp2 Proteins Puma Stat1 Stat3 Tbk1 Xrcc1 Xrcc3

Corresponding Organization :

Other organizations : University of Chinese Academy of Sciences, Shanghai Institute of Materia Medica, Shanghai Jiao Tong University, Ocean University of China, Shanghai Institute of Nutrition and Health

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Variable analysis

independent variables

Antibodies against the following proteins: γ-H2AX, CDT1, Chk1, p-Chk1, p-Chk2, caspase-3, caspase-7, caspase 9, PARP, Bak, BID, Puma, Noxa, Bcl-XL, XIAP, MRE11, c-IAP1, c-IAP2, STAT1, p-STAT1, STAT3, p-STAT3, STING, p-STING, TBK1, p-TBK1, IRF3, p-IRF3, p38, p-p38, ERK1/2, p-ERK1/2, MEK1/2, p-MEK1/2, JNK, Axin2, KU70, KU80, RPA32, p-RPA32, Bax, MCL1, PTIP, Chk2, PARP2, XRCC1, XRCC3, MLH1, MSH2, TNKS1/2, p-JNK, PARP1, PAR, RAD51, CTIP, MCRS1/MSP58, PALB2, MAD2L2/REV7, BRCA1, BRCA2

dependent variables

Levels of the proteins listed as independent variables

control variables

GAPDH

positive controls

Not specified

negative controls

Not specified

Annotations

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